In this proposal our current studies on the SV40 virus as a model for cellular chromatin are extended in two general directions: 1) Our recent discovery of multiply intertwined catenated SV40 dimers as intermediates in segregation of daughter minichromosomes (1) and parallel work by other groups on both prokaryotic and eukaryotic type II DNA topoisomerases (2-14) made the isolation of green monkey type II topoisomerase both an extremely important and feasible project. We will isolate, purify and characterize type II DNA topoisomerase(s) from both uninfected and SV40-infected green monkey cells, and will then use the enzyme(s) in a number of different approaches, in particular: (i) detailed comparison of the enzymes from uninfected and SV40-infected cells with an emphasis on possible participation of virus-coded polypeptides in the type II topoisomerase complex: (ii) studies on the location of the type II topoisomerase in the nuclei and its associations with other cellular poteins using both direct fractionation methods and immunochemical approaches. In particular, we will ask whether type II enzyme or a specific subset of its subunits can be found in association with isolated replicating minichromosomes and if so, what is its location in relation to the SV40 DNA sequence. 2) We will use a strongly refined version of the core nucleosome mapping approach to determine nucleosome arrangement(s) over the entire SV40 genome. Our previous work (15-17) has already shown that nucleosomes are distributed non-randomly at least with regard to a specific exposed about 400-bp region of the SV40 genome in a substantial proportion of the minichromosomes. Detailed information about the arrangement of nucleosomal cores in relation to SV40 DNA sequence should not only clarify the ways nucleosomes are distributed along a "linearized" nucleoprotein fiber but may also provide insights into the higher order folding pattern in the SV40 minichromosome. Since SV40 is an oncogenic virus, understanding of the fine structure and replication of SV40 chromatin will contribute not only to the chromatin and replication fields but to virology and oncology as well.